Stabilization of liquid solutions of recombinant protein for frozen storage

ABSTRACT

The invention relates to a method for stabilizing a bulk solution of recombinant protein for frozen storage, which comprises providing a partially-purified solution of recombinant protein which has a monovalent salt concentration of at least 100 mM, and adding a carbohydrate to said solution in an amount sufficient that, upon freezing, the solution has a glass transition temperature of −56° C. or higher.

RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 12/597,333, filed Oct. 23, 2009, issued as U.S. Pat. No.8,187,799 on May 29, 2012, which is a national stage entry (under 35U.S.C. §371) of International Application No. PCT/US2008/061147, filedApr. 22, 2008, which claims benefit of U.S. Provisional PatentApplication No. 60/926,698, filed Apr. 26, 2007. The entire disclosureof each of the foregoing patent applications is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The invention relates to the freezing and storage of liquid solutions ofrecombinant protein, preferably bulk solutions.

BACKGROUND OF THE INVENTION

Production of recombinant proteins in cell culture normally involves aseries of purification steps, by which the desired protein product isrecovered from recombinant host cells and/or the associated culturemedia. Many important recombinant proteins are produced on a largecommercial scale. In the case of pharmaceutical proteins, for example,it is not uncommon for more than one purification stage to be used toachieve the desired level of product purity.

It can be necessary to store a bulk solution of recombinant proteinwhich has been initially purified, but not finally purified, prior tofinal purification for formulation. For example, a protein-containingproduct of a recombinant fermentation reaction can be initially purifiedin an affinity or ion exchange column. After an initial pass through thecolumn, the protein product is only partially purified, and the solutionstill contains contaminants such as remnants of the cell culture andother proteins. Prior to final formulation into a pharmaceuticalproduct, the bulk solution must be further processed to obtain theprotein in a satisfactory purity.

Normally the solution, e.g. elution buffer, which is used to recover theprotein from a first-pass purification treatment is a high saltsolution. In the case of elution from a column, a high saltconcentration is needed to release the protein from the column.Accordingly, the “bulk” solution recovered from first pass purificationtreatment can comprise a solution having a high concentration ofmonovalent salts, normally sodium chloride but potentially potassiumchloride, or other salts.

The storage of a “bulk” solution of recombinant protein poses uniquechallenges due to the high salt concentration and very low proteinconcentration of the solution. Ideally, proteins are stored below theglass transition temperature to assure stability, since in the glassystate, protein inactivation and denaturation are extremely slow on apharmaceutical time scale. On the other hand, the presence of high saltconcentration in a solution tends to depress its glass transitiontemperature, and in solutions with high salt concentration and lowprotein concentration, very low temperatures are needed to achieve thisstate.

Frozen storage at higher temperature is desirable for bulk solutions inlarge volume quantities for cost and efficiency reasons, but whilepreserving the stability and activity of the protein.

SUMMARY OF THE INVENTION

The invention is a method for stabilizing a liquid solution ofrecombinant protein for frozen storage, which comprises: providing asolution of recombinant protein wherein said solution has a monovalentsalt concentration, e.g. of NaCl and/or KCl, of at least 100 mM; addinga carbohydrate to said solution in an amount sufficient to provide thesolution, upon freezing, with a glass transition temperature of −56 C orhigher; and freezing said solution for storage.

The invention also provides a liquid solution of recombinant proteinwhich is stabilized for frozen storage, which contains a carbohydrate inan amount sufficient to provide the solution, upon freezing, with aglass transition temperature of −56 C or higher

DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect on preserving recombinant protein activity ofadding carbohydrate, with or without other excipients as described inthe text, to a bulk solution of recombinant Factor VIII. The fourdifferent formulations are designated F1, F2, F3 and F4. Factor VIIIactivity was assayed after freezing and storage at −30° C., at differenttime points as shown, for up for 24 weeks. Before addition ofcarbohydrate, the bulk solution contained approximately 600 mM NaCl, 10mM CaCl₂, 20 mM imidazole and 0.1% Triton X-100, except F3 which wasdiluted as described herein. The graph plots time against coagulationpotency (IU/mL).

FIG. 2 shows loss of recombinant FVIII activity after freezing andstorage at −70° C. and −30° C. of the bulk Factor VIII solution used inthe experiments illustrated in FIG. 1 but without stabilizationexcipients. The designation “LN₂ to −70° C.” indicates that the sampleswere frozen in liquid nitrogen prior to storage at −70° C., and “LN₂ to−30° C.” indicates that the samples were frozen in liquid nitrogen priorto storage at −30° C. “EVA to −70° C.” indicates that the samples werefrozen in polymer storage bags and stored at −70° C.

DESCRIPTION OF PREFERRED EMBODIMENTS

The liquid solution of recombinant protein may comprise a solution ofany recombinant protein obtained from recombinant cell culture usingaffinity chromatography, ion-exchange chromatography, or the like. In apreferred embodiment, the solution is a bulk solution, which comprises asolution which has been partially purified. In all embodiments, theliquid solution is a high-salt solution, preferably an aqueous solution.

Recombinant proteins include, for example and without limitation,coagulation factors, virus antigens, bacterial antigens, fungalantigens, protozoal antigens, peptide hormones, chemokines, cytokines,growth factors, enzymes, blood proteins such as hemoglobin,α-1-antitrypsin, fibrinogen, human serum albumin, prothrombin/thrombin,antibodies, blood coagulation and/or clotting factors, and biologicallyactive fragments thereof; such as Factor V, Factor VI, Factor VII,Factor VIII and derivatives thereof such as B-domain deleted FVIII,Factor IX, Factor X, Factor XI, Factor XII, Factor XIII, FletcherFactor, Fitzgerald Factor, and von Willebrand Factor; milk proteins suchas casein, lactoferrin, lysozyme, α-1 anti-trypsin, protein factors,immune proteins, and biologically active fragments thereof; andantibodies, including monoclonal antibodies, single chain antibodies,antibody fragments, chimeric antibodies, humanized antibodies, and otherantibody variant molecules which can be produced in recombinant cellculture.

A currently preferred recombinant protein is recombinant Factor VIII.Factor VIII as used herein includes engineered variants of Factor VIII,such as B-domain deleted variants of Factor VIII.

A “bulk” solution within the meaning of the present invention comprisesa partially but not fully purified liquid solution of recombinantprotein, which contains at least 100 mM monovalent salt. The monovalentsalt is preferably NaCl which is commonly used to elute recombinantproteins from purification columns. However, the NaCl may be replaced,in whole or in part, with KCl. The bulk solution may also containvarying amounts of other salts, such as divalent salts including calciumchloride.

By “partially but not fully purified” is meant the liquid solution hasbeen subjected to at least one purification step, but the liquidsolution still contains sufficient residual impurities that at least onefurther purification step is required prior to final productformulation. For example, a “bulk” solution of recombinant Factor VIIImust be further purified prior to final formulation, which in the caseof Factor VIII and other proteins may include lyophilization.

The liquid solution contains at least 100 mM monovalent salt, preferably100 mM NaCl, more preferably at least 300 mM NaCl, more preferably atleast 500 mM NaCl, more preferably at least 560 mM NaCl and still morepreferably at least 600 mM NaCl. It is not uncommon for bulk solutionsof recombinant protein to have this high monovalent salt concentrationfollowing an initial purification stage.

In further embodiments of the invention, the liquid solution contains100-200 mM NaCl, 100-300 mM NaCl, 200-300 mM NaCl, 100-400 mM NaCl,100-500 mM NaCl, 100-600 mM NaCl, 100-800 mM NaCl, 300-500 mM NaCl,300-600 mM NaCl, 300-800 mM NaCl, 400-600 mM NaCl, 400-800 mM NaCl,500-600 mM NaCl, 560-700 mM NaCl and 500-800 mM NaCl.

The “bulk” solutions of the invention are further characterized by theirvery low protein concentration. In embodiments of the invention, theconcentration of recombinant protein in the bulk solution can be as lowas 0.0001 micromolar, 0.001 micromolar, or 0.01 micromolar. Inembodiments of the invention, the concentration of recombinant proteinin the bulk solution can be as high as 10 micromolar, 1 micromolar, or0.1 micromolar. Any concentration of protein falling within anycombination of these upper and lower limits is an embodiment of a “bulk”solution within the meaning of the invention.

The carbohydrate is added to the liquid solution, prior to freezing, inan amount sufficient to provide the solution, upon freezing, with aglass transition temperature of −56° C. or higher, more preferably atleast −34° C., or any temperature expressed by a whole or fractionalnumber therebetween. The normal glass transition temperature of a highsalt, low protein bulk solution is substantially less than −56° C., e.g.−60to −70° C. The amount of added carbohydrate needed to elevate theglass transition temperature to −56° C. should take into account, as onefactor, the protein concentration. Higher protein concentrations tend tothemselves elevate the glass transition temperature of a bulk solution.As other factors, the amount of carbohydrate should not excessivelyincrease the viscosity of the solution, and preferably the viscosity ismaintained below about 9.0 cP. The conductance of the solution can bechanged by carbohydrate addition, and preferably, should be maintainedbelow about 39 mS/cm.

Freezing the solution, in the context of the present invention, meansfreezing the bulk liquid solution, and is to be distinguished fromfreeze-drying, which involves different technical considerations.

The carbohydrate can be the type of carbohydrate normally used inpharmaceutical formulations, including sugars and di-, oligo- andpoly-saccharides. Examples include dextrans, cyclodextrans, chitosans,starches, halyuronic acids, cellulose, raffinose, maltose, lactose,stachyose, and combinations thereof. Preferred examples arecarbohydrates which are approved for injection, which includes sucrose,trehalose, hydroxyethylstarch, dextran, or combinations thereof.Pharmaceutical grade carbohydrates are available commercially from anumber of suppliers.

The precise amount of carbohydrate needed to protect the solution duringfreezing can be readily determined, for example by differential scanningcalorimetry, and depends on the particular protein and the particularcarbohydrate. Currently preferred amounts of carbohydrate are 8-25%(w/w) based on weight of liquid solution.

In specific embodiments of the invention, the amounts of carbohydrateare about 8-15%, 12-20%, 16-20%, 15-25%, and 20-25% (w/w) based onweight of liquid solution.

Other components from the initial purification (e.g. elution) may bepresent in a bulk solution, including a surfactant (e.g. Tween 80 orTriton-X), calcium chloride, or imidazole. Other excipients can be addedto the liquid solutions. As shown in the below formulations, additionalsurfactant may be added as an excipient. As a further excipient, anamino acid (e.g. glycine) may be added.

EXAMPLES

The invention is illustrated using, as exemplary recombinant protein,recombinant Factor VIII. Recombinant Factor VIII is produced usingmethods known in the art, for example as described in U.S. Pat. Nos.5,576,194; 5,804,420; and 5,733,873. In preferred embodiments,recombinant Factor VIII is produced in mammalian cells in large-scalefermentation reactors, in media which can be serum-free and/or proteinfree. Preferably the recombinant Factor VIII is secreted into the mediaby the recombinant cells.

Recombinant Factor VIII (full length) was expressed from host cells andpurified from clarified tissue culture fluid by membrane adsorberchromatography. The membrane adsorber process isolates and concentratesrecombinant Factor VIII from the tissue culture fluid by binding andelution (generally as described in Suck et al., J. Biotechnology, 121:361-367, 2006.) The eluate was divided into four batches and each batchwas transferred into a sterile bottle (400 mL in each bottle). Inaddition to recombinant Factor VIII and residual impurities whichremain, the eluate (bulk solution) contained approximately 600 mM NaCl,10 mM CaCl₂, 20 mM imidazole and 0.1% Triton X-100. The concentration ofrecombinant Factor VIII in the eluate was approximately 0.067micromolar.

A carbohydrate or combination of carbohydrates, along with otherexcipients as indicated, were then added to each bottle at roomtemperature in the amounts shown as Formulations I, 2, 3 and 4 in Table1 below.

TABLE 1 Formulation 1 Formulation 2 Formulation 3 Formulation 4 8%Sucrose 15% Sucrose 10% Hydroxyethyl 15% Dextran 3% Glycine starch  8%Trehalose  8% Trehalose 80 ppm Tween 80 ppm Tween

All components in Table 1 are shown in percent by weight based on weightof solution, Carbohydrates and other excipients were obtainedcommercially. Each fresh formulated batch was sampled. Samples fromFormulations (1), (2), (3) and (4) were assessed for Factor VIIIactivity using a standard coagulation assay.

Formulation 3 was prepared from the same eluate but was diluted with abuffer containing 20 mM imidazole and 10 mM CaCl₂ to decrease the NaClconcentration by half. This dilution was performed to examine theapplicability of the process of the invention to solution having alower, but still relatively high, monovalent salt concentration.

The glass transition temperature of each sample was determined usingDifferential Scanning calorimetry (DuPont Modulated DSC). The glasstransition temperatures exhibited by Formulations I, 2, 3 and 4 were,respectively, −56° C., −52.1° C., −34.9° C. and −35.5° C. In each case,the glass transition temperature is significantly higher than the glasstransition temperature observed in the absence of an added carbohydrate(which for the same bulk solution without carbohydrate was determined tobe between −60 and −70° C.). The viscosities of the formulations were:Formulation 1: 1.8428 cP; Formulation 2: 3.1089 cP; Formulation 3:6.8076 cP; and Formulation 4: 7.2123 cP. The conductivities of theformulations were: Formulation 1: 27.8 mS/cm; Formulation 2: 25.57mS/cm; Formulation 3: 21.05 mS/cm; and Formulation 4: 32.1 mS/cm.

All of the formulations were found to be stable after frozen storage at−80, −30 −18 and −14° C. up to 24 weeks, as determined by coagulationassay for Factor VIII activity at various time points, withoutsignificant loss of activity.

As shown in FIG. 1, all four formulations in accordance with theinvention maintained Factor VIII coagulation activity at substantiallythe initial level after storage at −30° C. for up to 24 weeks.

As shown in FIG. 2, in the absence of the added excipients, the solutionlost substantially all of its Factor VIII coagulation activity afteronly 1 day of storage at −30° C.

What is claimed is:
 1. A liquid solution of recombinant protein which isstabilized for frozen storage, and which has an NaCl and/or KClconcentration of at least 100 mM, which contains a carbohydrate in anamount sufficient to provide the solution, upon freezing, with a glasstransition temperature of −56° C. or higher, wherein the carbohydrate ispresent in an amount of 8-25% by weight of the solution.
 2. The liquidsolution of recombinant protein of claim 1, wherein the solution is abulk solution.
 3. The liquid solution of recombinant protein of claim 1,wherein the solution has an NaCl concentration of at least 100 mM. 4.The liquid solution of recombinant protein of claim 1, wherein thecarbohydrate is selected from the group consisting of sucrose,trehalose, hydroxyethylstarch, dextran, and combinations thereof.
 5. Theliquid solution of recombinant protein of claim 1, wherein the liquidsolution has an NaCl concentration of at least 300 mM.
 6. The liquidsolution of recombinant protein of claim 1, wherein the liquid solutionhas an NaCl concentration of at least 600 mM.
 7. The liquid solution ofrecombinant protein of claim 1, wherein the recombinant protein isFactor VIII.
 8. The liquid solution of recombinant protein of claim 1,wherein the glass transition temperature is between −56° C. and −35° C.9. A liquid solution of recombinant Factor VIII, which contains an NaClconcentration of at least 100 mM, contains recombinant Factor VIII at aconcentration between 0.0001 micromolar and 10 micromolar, and isstabilized for frozen storage by addition of a carbohydrate in an amountsufficient to provide the solution, upon freezing, with a glasstransition temperature of −56° C. or higher, wherein the carbohydrate ispresent in an amount of 8-25% by weight of the solution.
 10. The liquidsolution of recombinant Factor VIII of claim 9, wherein the solution isa bulk solution, the NaCl concentration is at least 300 mM, and theFactor VIII concentration is between 0.001 micromolar and 1.0micromolar.
 11. The liquid solution of recombinant protein of claim 5,wherein the solution is a bulk solution.
 12. The liquid solution ofrecombinant protein of claim 5, wherein the liquid solution has an NaClconcentration of at least 600 mM.
 13. The liquid solution of recombinantprotein of claim 5, wherein the carbohydrate is selected from the groupconsisting of sucrose, trehalose, hydroxyethylstarch, dextran, andcombinations thereof.
 14. The liquid solution of recombinant protein ofclaim 5, wherein the recombinant protein is Factor VIII.
 15. The liquidsolution of recombinant protein of claim 5, wherein the glass transitiontemperature is between −56° C. and −35° C.